Stability of a protein is very often the critical factor which imposes a limit on practical use of that protein in technological or medical applications. Therefore, much effort has been directed into understanding of those processes that lead to loss of the biological activity. These processes can conveniently be divided into covalent and conformational processes as described by Ahern, T.J., and Klibanov, A.M. in Protein Structure, Folding and Design, (Oxender, D.L., ed. 1986) Alan R. Liss, Inc. New York, pp. 283. The covalent processes include deamidation of asparagine, destruction of disulfide bridges and cleavage of the peptide bonds at aspartic acid residues, whereas conformational processes include changes in the spatial structure of the polypeptide backbone. Stabilized proteins are produced by methods which are able to diminish covalent and/or conformational processes responsible for the inactivation.
There are two general ways to prepare a protein more stable than the original one. By genetic engineering it is possible to exchange one amino acid with the other which is less susceptible to a reaction deleterious for stability or with a new amino acid which contributes to the stabilizing forces. By chemical cross-linking of the original protein it is possible to introduce additional covalent links, which then stabilize the active conformation. There are many reports showing that stabilized proteins can be obtained by both approaches. With respect to the present invention, it should be noted that one of the most widely used protein cross-linking reagents is glutaraldehyde, which is an efficient cross-linker because it always contains polymeric aldehydes. See Peters, K., and Richards, F.M. 46 Ann. Rev. Biochem. 523 (1977).
Glycoproteins are proteins that contain covalently linked sugar chains. The carbohydrate chains are usually not directly involved in biological activity of a glycoprotein, and particularly not in enzymic activity of glycoenzymes. See Tarentino, A.L., Plumer, T.H., and Maley, F. 249 J. Biol. Chem. 818 (1974); Chu, F.K., Trimble, R.B., and Maley, F. 253 J. Biol. Chem. 818 (1978); and Barbaric, S., Mrsa, V., Ries, B., and Mildner, P. 234 Arch. Biochem. Biophys. 567 (1984). We have shown that glycoenzymes can be specifically cross-linked through their carbohydrate chains Kozulic, B., Barbaric, S., Ries, B., and Mildner, P. 122 Biochem. Biophys. Res. Commun. 1083 (1984). Our cross-linking procedure Kozulic, B., Barbaric, S., Ries, B., and Mildner, P. 122 Biochem. Biophys. Res. Commun. 1083 (1984) consisted of two steps. In the first step, susceptible monosaccharides were oxidized by periodate. This resulted in aldehyde groups which in the second step reacted with a bifunctional cross-linker, such as adipic acid dihydrazide.
As demonstrated by electrophoresis, cross-linking with the dihydrazide produces intramolecularly and intermolecularly cross-linked derivatives. See Kozulic, B., Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S. 15 Appl. Biochem. Biotech. 265 (1987). We have also shown that the cross-linking of carbohydrate chains improves greatly the stability of a glycoenzyme, most likely by increasing the rigidity of its polypeptide backbone. See Kozulic, B., Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S. 15 Appl. Biochem. Biotech. 265 (1987).
As a control, in that study we have also examined whether intermolecularly cross-linked oligomers were in part the result of Schiff base formation between sugar aldehyde groups and protein amino groups. The results clearly showed that the intermolecularly cross-linked oligomers are mostly the result of adipic acid dihydrazide reaction, although oxidized invertase and acid phosphatase, but not glucose oxidase, without the cross-linker formed a very low amount of oligomers. See Kozulic, B., Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S. 15 Appl. Biochem. Biotech. 265 (1987). We have also shown that the stabilization effect observed is a result of the cross-linking reaction, since the oxidized invertase and glucose oxidase were essentially as stable as the native enzymes, while oxidized acid phosphatase was less stable than the native enzyme. See Kozulic, B., Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S. 15 Appl. Biochem. Biotech. 265 (1987). Such results appeared reasonable, since the presumed linkage (Schiff base) between the oxidized sugar and the protein part is reversible. Accordingly, at that time the possibility of glycoprotein stabilization only by periodate oxidation was regarded as inapplicable. This assumption was supported by the results in Woodward, J. and Wiseman, A. 29 J. Chem. Tech. Biotechnol. 122-126 (1979), wherein oxidized invertase showed no increase in stability.
Woodward and Wiseman, in their paper on stabilization of invertases, considered the possibility of Schiff base formation but, as well as Kozulic et al Kozulic, B., Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S. 15 Appl. Biochem. Biotech. 265-278 (1987) they concluded (page 125, first paragraph): "It is assumed that these aldehyde groups of the mannan moiety did not cross-link with amino groups of the protein moiety since there was no effect on thermal stability." Their experimental data clearly showed (Table 1) that the inactivation constant of the native and oxidized invertase was identical (0.22 min.sup.-1).
Accordingly, there is no indication in the prior art that periodate oxidation followed by an incubation of the oxidized glycoprotein can result in better stability, and therefore the 10-fold increase in stability now discovered by the applicants was totally unexpected.
A question arises as to how the findings of the present invention could be reconciled with the experimental data in the prior art discussed above. In relation to Applicants' own work, in Kozulic, B. Leustek, I., Pavlovic, B., Mildner, P., and Barbaric, S., 15 Appl. Biochem. Biotech. 265-278 (1987) we did not investigate in detail the properties of fully oxidized glycoenzymes because they formed partially insoluble polymers after addition of adipic acid dihydrazide. Concerning the work of Woodward and Wiseman, after careful reading of their experimental procedures (page 123, paragraph 2.2.5.) Applicants have found an important difference which explains the discrepancy in the experimental results. Specifically, (paragraph 2.2.5. lines 2-3), Woodward et al stopped the periodate oxidation reaction by addition of ethylene glycol. Periodate reacts with ethylene glycol to form formaldehyde. As one of the most reactive aldehydes, formaldehyde reacts with protein amino groups to form an imine which can further react with another amino group: EQU R--NH.sub.2 +HCHO.dbd.R--N=CH.sub.2 EQU R--N.dbd.CH.sub.2 +R'--NH.sub.2 .fwdarw.R--NHCH.sub.2 NH--R'
Since this reaction was allowed to proceed 1 hour (paragraph 2.2.5. line 3), there was most likely no free amino group left to react with the aldehyde groups formed in the mannan moiety. Therefore, no stabilization effect could be observed.
From the above discussion it is clear that a choice of correct experimental conditions is of utmost importance, because apparently minor modifications can cause a profound effect. This situation is not uncommon, as exemplified by a case where a small change in reaction conditions caused a significant effect. Accordingly, both Thorpe et al. and Casellas et al. U.S. Pat. No. 4,911,912 have treated ricin with periodate in the presence of sodium cyanoborohydride, but Casellas et al have obtained unexpectedly better results when, instead of the whole molecule, only the ricin A chain was treated at a higher pH for a longer time ('912 column 23 and 24).
However, after full apprehension of the idea that an oxidized carbohydrate chain of a glycoprotein may act as an efficient intramolecular cross-linker due to its polyaldehyde character, we have decided to perform more detailed experiments. Unexpectedly, they indicated that certain irreversible linkages were formed after a longer incubation of strongly oxidized glycoproteins. In addition, the new derivatives were found to be much more stable than the native enzyme.